Fig. 1

Necroptosis-dependent transcriptional hyperactivation. (A-I) All experiments were used with HT-29 colon cancer cells which is well established for necroptosis pathway. (A) RNA-seq-based expression of 163 genes in HT-29 cells treated with TNF-α or TNF-α + SMAC + zVAD (herein, TSZ). (B) Scatter plot comparing the expression of TNF-α- and TSZ-inducible genes (log2 scale) in the TNF-α /DMSO RNA-seq (X-axis) versus the TSZ/DMSO RNA-seq (Y-axis). HT-29 cells were treated with TNF-α or TSZ. (C) Metagene representation of the ChIP-seq signal for p65 across 497 regions and 163 gene-associated peaks in HT-29 cells treated with TNF-α (30 ng/ml) or TSZ (TNF-α (30 ng/ml) + SMAC (200 nM) + zVAD (20 μM), hereafter referred to as TSZ for 4 h. Metagenes centered on the middle of 497 regions and 10 Kb around the center of 497 regions are displayed. (D) ChIP-seq profiles of p65 in HT-29 cells treated with TNF-α or TSZ for 4 h at the TNFAIP3, CSF1, and IL32 loci. (E) Metagene representation of the ChIP-seq signal for p65 across 497 regions in HT-29 cells treated with TNF-α or TSZ for 30 min or 4 h. Metagenes centered on the middle of 497 regions and 10 Kb around the center of 497 regions are displayed. (F) Western blot analysis of the nuclear and cytosol fractions. HT-29 cells were pretreated with necrostatin-1 (Nec-1, 40 μM) for 1 h and with TSZ for 30 min and 4 h. (G) HT-29 cells were treated with TSZ for the indicated periods, and IL-8 and IL-1β mRNA levels were measured by qPCR. (H) HT-29 cells were pretreated with Nec-1 and GSK’872 (10 μM) for 1 h and with TSZ for 4 h. IL-8, IL-1β, and CXCL1 mRNA levels were measured by qPCR. (I) HT-29 cells were pretreated with Nec-1 and GSK’872 for 1 h and with TSZ or TNF for 4 h. Cell lysates were analyzed by western blotting